Two distinct binding modes provide the RNA-binding protein RbFox with extraordinary sequence specificity

Specificity of RNA-binding proteins for target sequences varies considerably. Yet, it is not understood how certain few proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two distinct binding modes. The first exclusively accommodates cognate and closely related RNAs with high affinity. The second mode accommodates all other RNAs with reduced affinity by imposing large thermodynamic penalties on non-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA-binding site. Distinct binding modes by a single RNA-binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation in RNA-protein interactions.


Supplementary Figure 2. Correlation between HiTS-Eq replicates.
Correlation between the two matched replicates for all 16,384 7-mer RNA sequence variants at different RbFox concentrations (line: diagonal, y = x; R 2 , correlation coefficient). Source data are provided as a Source Data file. a Global affinity distributions for six RBPs (RbFox, PUM1, DAZ3, RBM6, SRSF8, and SRSF11) from HiTS-Eq data and ENCODE RBNS datasets. b Affinity ratios between the highest affinity value to the median affinity value for the six RBPs. Source data are provided as a Source Data file.

Supplementary Figure 4. RbFox binding to 5-mer RNA variants.
a Distribution of relative association constants (KA,rel) of RbFox for all 1,024 5-mer RNA sequence variants (red triangle: reference GCAUG; green triangle: GCACG). b Correlation between relative association constants (KA,rel) from the HiTS-Eq measurements and R value from RNA Bind-n-Seq (RBNS) measurements for all 1,024 5-mer RNA sequence variants. (line: diagonal, y = x; R 2 , correlation coefficient). c Linear coefficient for -1 and +1 nucleotide position of all 1,024 5-mer RNA sequence variants calculated with the PWM binding model (negative values: destabilization). Source data are provided as a Source Data file.

Supplementary Figure 5. Analysis of the binding affinities to RbFox for 5'-GCNUG sequences.
Relative affinities (KA,rel) for selected 5-mer RNA variants, indicated on the below. 48 KA,rel values correspond to all 5-mer with 7 randomized nucleotides (vertical line: median; box: variability through lower quartile and upper quartile; whiskers: variability outside the lower and upper quartiles). Source data are provided as a Source Data file. Figure 6. Analysis of RbFox binding to 7-mer RNA variants with quantitative binding models.

Supplementary
a Correlation between experimental KA,rel values for RbFox for each 7-mer with values calculated with the PWM binding model; (red dots: consensus 5-mer; line: diagonal, y = x; R 2 : correlation coefficient). b Linear coefficient for each nucleotide position calculated with the PWM binding model (negative values: destabilization). c Correlation between experimental KA,rel values for RbFox mut for each 7-mer with values calculated with the PWC binding model (red dots: consensus 5-mer; line: diagonal, y = x; R 2 : correlation coefficient). d Linear coefficients for each pairwise coupling between all nucleotides calculated with the PWC model; (black frames: couplings in consensus 7-mer, negative values: destabilization). Source data are provided as a Source Data file.

Supplementary Figure 7. Correlation between HiTS-Eq replicates for RbFox mut .
Correlation between the two matched replicates for all 16,384 7-mer RNA sequence variants at different RbFox mut concentrations; (line: diagonal, y = x; R 2 , correlation coefficient). Source data are provided as a Source Data file.

Supplementary Figure 8. RbFox mut binding to 5-mer RNA variants.
a Affinity distribution (KA,rel) of RbFox mut for all 5-mer RNA sequence variants (blue); (bin size: 100). For reference, the affinity distribution of wild type RbFox (grey) is provided as well. b Linear coefficient for -1 and +1 nucleotide position of all 1,024 5-mer RNA sequence variants calculated with the PWM binding model (negative values: destabilization). Source data are provided as a Source Data file. a Superposition of 1 H -15 N HSQC spectra obtained with 15 N-RbFox RRM and increasing amount of 5'-UGCAUGU RNA. The peaks corresponding to the free and RNA-bound RRMs (RRM:RNA ratios of 1:0, 1:0.1, 1:0.2, 1:0.5 and 1:1) are colored as black, red, orange, green and blue, respectively. b Superposition of 1 H -15 N HSQC spectra obtained with 15 N-RbFox RRM and increasing amount of 5'-UGCAUAU RNA. The color scheme is the same as in a. c Superposition of 1 H -15 N HSQC spectra obtained with 15 N-RbFox RRM and increasing amount of 5'-UUCAUGU RNA. The peaks corresponding to the free and RNA-bound RRMs (RRM:RNA ratios of 1:0, 1:0.4, 1:1.2, and 1:2) are colored as black, green, blue and red, respectively. Figure 11. Comparison of the surface electrostatics of the two RbFox-RNA complexes.

Supplementary
a Ribbon diagrams of the superposed RbFox structures bound to the different RNA (blue: complex with 5'-UGCAUGU; orange: complex with 5'-UGCAUAU). The side chain of the flipped out E164 residue which alters surface electrostatics through its relocation is explicitly labeled. b,c Surface electrostatics for the two RbFox-RNA complexes with 5'-UGCAUGU and 5'-UGCAUAU, respectively.